Rickettsia are intracellular pathogenic bacteria responsible for various diseases on Humans and animals. Rickettsia are transmitted by arthropods, most frequently ticks, lice and mites, and cause major illnesses such as epidemic typhus or Rocky Mountain spotted fever. The genus Ehrlichia comprises several species pathogenic for humans and mammals such as E. chaffeensis, responsible for Human monocytic ehrlichiosis, E. canis, the causing agent of canine monocytic ehrlichiosis, or E. phagocytophillia, the agent of Human granulocytic ehrlichiosis.
Another species, Ehrlichia ruminantium, formerly known as Cowdria ruminantium, is the causing agent of heartwater or cowdriosis, an economically important disease of domestic ruminants. Heartwater can cause up to 80% mortality in susceptible animals. E. ruminantium is transmitted by Amblyomma ticks and is present in Sub-Saharan Africa and surrounding islands, including Madagascar. Heartwater is also present in several Caribbean islands and is threatening the American mainland.
Serological diagnostic tests of heartwater using crude antigens from whole bacteria detect false positive reactions due to common antigenic determinants. ELISA-based and serological diagnostics have been developed using the Map 1 (WO 9914233; Sumption et al. Clin Diagn Lab Immunol. 10: 910-916, 2003) and the GroEL (WO 9914233) antigens. Other peptides for serological diagnostic have been described (US 2002004051, US 20020132789, WO 02/066652). Although they have dramatically improved specificity, they still display cross reaction with E. canis and E. chaffeensis. Furthermore, the life span of anti-Map 1 antibodies is rather short.
PCR-based diagnostic methods represent methods of choice for the sensitive and specific detection of Ehrlichia in clinically reactive or asymptomatic carrier ruminants, as well as in vectors. However, in the field, hosts and vectors can be co-infested by several parasites and the diversity of pathogen species is further complicated by the existence of extensive intra-species diversity. Improved methods are required to discriminate between strains of differing pathogenesis.
Vaccination against heartwater has long been based on “infection and treatment”. Naïve animals are inoculated with blood containing virulent organisms, a procedure which carries a high risk of uncontrolled clinical reactions and the inadvertent spread of undesirable parasites and viruses. A first generation of cowdriosis inactivated vaccine based on cell-cultured derived elementary bodies was developed. Although representing a considerable improvement and the first heartwater vaccine acceptable for widespread use, the level of protection conferred is still not fully satisfactory. Indeed, all animals develop a clinical reaction at challenge despite vaccination. Furthermore, livestock also faces challenge by genetically and antigenically diverse strains.
Diversity of E. ruminantium is a key problem which has been recognized for a long time, but insufficient information is available for optimum vaccine formulation and specific diagnostic. The diversity of E. ruminantium was demonstrated at the antigenic level by cross-immunisation studies. Variable antigens were identified by ELISA and immunoblot using cross-absorbed immune sera. Genetic diversity was later demonstrated when sequencing the Map 1 gene which showed a high degree of sequence heterogeneity concentrated in three hypervariable regions. This DNA polymorphism was shown to correlate with antigenic polymorphism. Genomic polymorphism was also detected using RAPD and RFLP markers. The map1 gene initially considered as a good marker for geographic diversity, was recently shown not to be geographically constrained. Furthermore, there is no evidence of evolution of map1 under positive selection pressure. Map1 was therefore reported as not being important for evasion of host immune response.